Peptide:MHC (pMHC) tetramers are reagents that can identify T cells based directly on their antigen receptor specificity. Recent developments in the generation and use of these reagents have made them a powerful new tool with which to study antigen-specific T cell responses, particularly in humans where experimental manipulation of T cells is extremely limited by feasibility issues. The overall objective of the Tetramer Core is to provide researchers from each of the proposed projects with the resources necessary to use tetramer technology to study allergen-specific T cells in mice and humans. The specific aims of the Tetramer Core will be 1) to generate pMHCII tetramers to defined allergens studied in the proposed projects, and 2) to provide technical expertise in tetramer-based enrichment techniques to enable identification of low frequency allergen-specific T cells. State-of-the-art tetramers will be generated incorporating the most advanced technical refinements to date. Recombinant DNA constructs encoding soluble forms of pMHCII molecules fused to immunogenic peptide epitopes from defined allergens will be cloned and expressed in an insect cell expression system. Purified biotinylated pMHCII complexes will then be tetramerized via their interaction with fluorescently labeled streptavidin molecules. For each allergen studied in human subjects, a panel of tetramers corresponding to several common HLA-DR alleles will be generated to ensure adequate applicability of these reagents to the HLA-diverse study population. Because antigen-specific T cell populations are often present in very low frequencies, tetramers will often be used in conjunction with antibody-coated magnetic beads to enable enrichment of tetramer-positive cells. This will allow for high-resolution detection of allergen-specific T cells from small amounts of human cell samples. The Tetramer Core will provide technical support to researchers so they can quickly master these techniques. Collectively, the services provided by the Tetramer Core will enable researchers to immediately take advantage of this powerful technology that otherwise may not be readily available at a practical scale for many years.